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A kind of biotransformation preparation method of steroid intermediate

A biotransformation and intermediate technology, applied in the field of biotransformation preparation of steroid intermediates, can solve the problems of unfavorable transformation methods for industrialization promotion, limited use of organic solvents, loss of substrate conversion ability, etc., to achieve increased conversion rate, The effect of increasing permeability and increasing speed

Active Publication Date: 2021-07-30
JIANGSU YUANDA XIANLE PHARMA
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] C commonly used on steroids 11 The organic solvents for α-hydroxylation fermentation include methanol, ethanol, propanol, propylene glycol, etc. Although the use of these solvents will improve the conversion efficiency to a certain extent, the organic solvent itself is poisonous to microorganisms. Contact will cause microbial cells to age rapidly and lose their ability to transform substrates, which means that the amount of organic solvent used is limited, and the process will naturally encounter bottlenecks when increasing the feed concentration
At the same time, the use of organic solvents will also bring serious safety and environmental protection problems. Now that the requirements for safety and environmental protection are becoming more and more stringent, it is not conducive to the industrialization of transformation methods.
[0006] Publication number is that the Chinese patent of CN103146793B reports a kind of C that adds biocatalyst 11 α-Hydroxylation biotransformation method, but still adopts the method of dissolving the substrate in ethanol, the maximum concentration of the substrate during the transformation process is only 8g / L, and the highest biotransformation rate is only 80%

Method used

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  • A kind of biotransformation preparation method of steroid intermediate
  • A kind of biotransformation preparation method of steroid intermediate
  • A kind of biotransformation preparation method of steroid intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 different feeding modes conversion and yield situation contrast

[0043] The present embodiment adopts the genus Metarhizium anisopliae as the fermentation strain, and the debrominated substance (intermediate I) of dexamethasone is the substrate, and its C 11 Bits are transformed while hydrolyzing C 21 Acetate, to obtain a C 11 α-hydroxyl, C 21 Hydroxyl-positioned steroid hormones important intermediate dexamethasone hydroxylate (intermediate Ⅱ). In this example, different feeding methods or different feeding concentrations were used for different shake flasks, so as to compare the conversion rate and yield.

[0044] The concrete steps of this embodiment are as follows:

[0045] (1) Bacteria culture

[0046] Transplant and culture Metarhizium anisopliae of Scarab on PDA slant medium, control the temperature at 28±1°C, cultivate for 5 days to obtain slant strains, and set aside;

[0047] Ratio of seed medium: yeast extract powder 10g / L, glucose 10g / L, ...

Embodiment 2

[0080] In this example, Metarhizium anisopliae was used as the fermentation strain, and dexamethasone debrominated product (intermediate I) was used as the substrate to prepare dexamethasone hydroxylate (intermediate II) by mold fermentation.

[0081] In this example, three different feeding methods are investigated.

[0082] The first feeding method: After the surfactant aqueous solution is sterilized, the steroid is added for emulsification to prepare a steroid suspension; then it is put into the pre-culture solution for fermentation and transformation.

[0083] The second feeding method: after the surfactant aqueous solution is sterilized, steroids are added for emulsification to prepare a steroid suspension; the supplementary nitrogen source material is sterilized separately to obtain a nitrogen source suspension; when feeding, the steroid is mixed with The suspension and the nitrogen source suspension are simultaneously added to the pre-culture solution for fermentation a...

Embodiment 3

[0112] The present embodiment provides a kind of biotransformation preparation method of steroidal intermediate, adopts Rhizopus stoloniferus as fermentation strain, with betamethasone debrominated (intermediate I) as substrate, to C 11 Bits are transformed while hydrolyzing C 21 Acetate, to obtain a C 11 α-hydroxyl, C 21 Hydroxyl-position steroid hormones important intermediate betamethasone hydroxylate (intermediate Ⅱ), the reaction principle is as follows figure 1 shown.

[0113] The concrete steps of this embodiment are as follows:

[0114] (1) Bacteria culture

[0115] Transplant Rhizopus stolonifera on the PDA slant medium for cultivation, control the temperature at 28±1°C, cultivate for 5 days to obtain slant strains, and set aside;

[0116] Prepare the seed medium, the ratio is: yeast extract powder is 10g / L, glucose is 10g / L, KH 2 PO 4 1g / L, KNO 3 It is 0.5g / L. After the preparation, it is divided into 200mL / shake flask, cooled to room temperature after steril...

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Abstract

The invention relates to a biotransformation preparation method of a steroid intermediate, belonging to the technical field of biopharmaceuticals. The present invention first prepares the steroidal suspension of intermediate I, then puts the suspension into the fermentation medium containing mold seeds, and C occurs under the action of mold 11 Hydroxylation and hydrolysis of C at the same time 21 Acetate, to obtain intermediate II. The invention adopts the emulsification feeding method to replace the operation of organic solvent dissolving substrate feeding, the concentration of feeding substrate can be increased to more than 15g / L, and the biotransformation rate can be increased to more than 90%. By adopting the method, adding nitrogen source nutrients while emulsifying, the biotransformation is more thorough under the condition of increasing the substrate feeding concentration, and the production cost is significantly reduced.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to a biotransformation preparation method of a steroid intermediate. Background technique [0002] Steroid hormones are the main components of corticosteroids, and betamethasone and dexamethasone are all steroid hormones, which have pharmacological effects such as anti-inflammation, anti-infection, anti-shock, and immune suppression. Steroidal C 11 α-hydroxylation is an indispensable step in the synthesis of various corticosteroids. Compared with chemical synthesis methods, biotransformation has outstanding advantages such as high conversion efficiency and good specificity, so the current steroid C 11 α-Hydroxylation is usually accomplished by microorganisms through a fermentation process. [0003] The enzymes that microorganisms catalyze the hydroxylation reaction are intracellular enzymes. Steroid substrate crystals must first be dispersed and gradually dissolved in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/08C12P39/00C12R1/645C12R1/845C12R1/66
CPCC12P33/08C12P39/00
Inventor 杜艳刘辉杜江涛王鹏辉单东奇白傲雪刘娟玲
Owner JIANGSU YUANDA XIANLE PHARMA
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